This invention relates generally to identification of markers and therapeutic targets in breast cancer.
1. Background
Breast cancer is one of the most common malignant diseases with about a million new cases per year worldwide. Despite use of a number of histochemical, genetic, and immunological markers, clinicians still have a difficult time predicting which tumors will metastasize to other organs. Some patients are in need of adjuvant therapy to prevent recurrence and metastasis and others are not. However, distinguishing between these subpopulations of patients is not straightforward, and course of treatment is not easily charted. There is a need in the art for new markers for distinguishing between tumors which will or have metastasized and those which are less likely to metastasize.
The majority of epithelial cancers originate due to altered gene expression leading to defects in cell differentiation and proliferation. One major mechanism of metastasis in breast carcinomas seems to be the so-called epithelial to mesenchymal transition (EMT) observed in invasive embryonic epithelia (Hendrix (1997) Am. J. Pathol. 150: 483-495; Pulyaeva (1997) Clin. Exp. Metastasis 15:111-120; McCormick (1989) Cancer Research 49: 4258-4263; Birchmeier (1996) Acta Anat. 156:217-226). One landmark of the EMT is the observation that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive. (e.g. MDA-MB-231, MDA-MB435 Hendrix (1997) Am. J. Pathol. 150: 483-495) whereas VIM negative cell lines (e.g. MCF-7, MDA-MB-468, MDA-MB-361)) are poorly invasive in nude mice. However, the identity of the regulators of EMT have remained largely unknown.
Prostate cancer is another cancer of considerable importance and in need of additional therapies and diagnostics. Prostate cancer is the most frequent solid cancer in older men and is a major cause of cancer-related death. Initially, prostate cancer involves androgen-dependent proliferation and differentiation of normal prostate basal cells of the prostate epithelia into secretory luminal epithelial cells; however, prostate cancer cells, ultimately become androgen-independent and resistant to hormone therapy. The prostate-specific antigen (PSA) gene has been used as a diagnostic indicator for androgen-independent and androgen-dependent prostate cancer (Ablin (1997) i J. Cancer Res. Clin. Oncol. 123:583-594). Expression of PSA in normal and cancerous luminal epithelial cells of the prostate is under the control of androgens acting through the androgen receptor. Thus, androgen independent prostate cancer generally exhibit decreased PSA expression. Unfortunately, PSA is expressed at least to some degree even in hormone-refractory prostate cancers, indicating that regulation of PSA expression involves an androgen-independent component as well (Sadar (1999) J. Biol. Chem. 274:7777-7783).
The ETS domain, which facilitates DNA binding, is the hallmark of a family of eukaryotic transcription factors. Members of the ETS domain family were originally identified on the basis of a region of primary sequence homology with the protein product of the v-ets oncogene encoded by the E26 avian erythroblastosis virus. ETS-domain transcription factors can be further subclassified primarily because of the high amino acid conservation in their ETS-domain, as well as the conservation of other domains or motifs, e.g., other sequences that can modulate the biological specificity of the protein. The ETS DNA binding domain is also conserved at the structural level, and is a divergent member of the winged helix-turn-helix superfamily of DNA binding proteins.
ETS-domain proteins act either as transcription activators or repressors, which activities are often regulated by signal transduction pathways such as the MAP kinase pathways. Many of the ETS-domain proteins are targeted to promoters by a combination of specific DNA-protein and protein-protein interactions. Many ETS-domain proteins are targets of signal transduction pathways, and are activated in response to various extracellular stimuli. Ets genes and their encoded proteins are involved in a variety of essential biological processes including cell proliferation and differentiation during embryonic development and in the adult. For a review of the ETS-domain transcription factor family, see, e.g., Sharrocks et al. (1997) Int. J. Biochem. Cell Biol. 29:1371-87.
In addition to their roles in developmental pathways, ETS-domain transcription factors have also been implicated in tumorigenesis. For example, a single ETS-related transcription factor, E1AF confers an invasive phenotype upon human cancer cells (Kaya et al. (1996) Oncogene 12:221-7). The role of E1AF as an activator of tumorigenesis is further supported by the finding that transfection of antisense E1AF into oral squamous cell carcinoma cells inhibits tumor invasion by down-regulating matrix metalloproteinase (MMP) genes, which are implicated in invasion and metastasis of tumor cells (Hilda et al. (1997) Oral Oncol. 33(6):426-30). Moreover, expression of E1AF in the nonmetastatic cell line MCF-7, an adenocarcinoma; of mammary breast, confers an invasive phenotype upon this cell line. (Kaya et al., 1996, supra)
Other ETS-domain containing proteins have also been implicated in tumorigenesis. For example, the Ets-1 and Ets-2 transcription factors, activate the promoter for invasion-associated urokinase and collagenase genes in response to epidermal growth factor (Watabe et al. (1998) Int J Cancer. 77:128-37). Furthermore, a putative ETS-related transcription factor interacts with a ras-activated enhancer in the mouse osteopontin promoter, a promoter whose expression correlates with the metastatic potential of cells (Guo et al. (1995) Mol Cell Biol 15:476-87).
Although several ETS-domain proteins have been identified (see, e.g., GenBank Accession No. AF071538), their precise biological roles, e.g., the role of such genes in oncogenesis, have not been identified. The present invention identifies the association of expression of one such ETS-domain protein with development of the metastatic phenotype.
Literature
The full-length DNA and amino acid sequences of human PDEF is described in GenBank Accession No. AF071538, and is entitled xe2x80x9cIsolation and characterization of a novel prostate epithelium-specific Ets transcription factor, PDEF.xe2x80x9d
Transfer of the metastatic phenotype by somatic cell fusion with the highly metastatic amelanotic C8161 human melanoma line is described by Barsky et al. (1997) xe2x80x9cEvidence of a dominant transcriptional pathway which regulates an undifferentiated and complete metastatic phenotype.xe2x80x9d Oncogene 15:2077-91.
Evidence suggesting malignant melanoma metastasis-regulatory gene may be on human chromosome 6 is described Welch et al. (1994) xe2x80x9cMicrocell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity,xe2x80x9d Oncogene 9:255-62. Loss of heterozygosity on the long arm of chromosome 6 in breast cancer is described in Noviello et al. (1996) xe2x80x9cLoss of heterozygosity on the long arm of chromosome 6 in breast cancer: possibly four regions of deletion.xe2x80x9d Clin Cancer Res. 2:1601-6. Suppression of human melanoma metastasis by introduction of chromosome 6 is described in You et al. (1995) xe2x80x9cSuppression of human melanoma metastasis by introduction of chromosome 6 may be partially due to inhibition of motility, but not to inhibition of invasion.xe2x80x9d Biochem Biophys Res Commun. 208:476-84; and Miele et al. (1997) xe2x80x9cSuppression of human melanoma metastasis following introduction of chromosome 6 is independent of NME1 (Nm23).xe2x80x9d Clin Exp Metastasis. 15:259-65.
For a review of the ETS-domain transcription factor family, see Sharrocks et al. (1997) xe2x80x9cThe ETS-domain transcription factor family,xe2x80x9d Int. J. Biochem. Cell Biol. 29:1371-87.
The full-length DNA and amino acid sequences of human ESX (SEQ ID NOS:4 and 5) are described in Chang et al. (1997) xe2x80x9cESX: a structurally unique Ets overexpressed early during human breast tumorigenesis,xe2x80x9d Oncogene 14:1617-22 and GenBank accession no. U66894.
The invention features methods for detection of metastatic and potentially metastatic cancerous cells by detection of expression of a gland-specific Ets transcription factor (GSEF) sequence, which encodes an Ets-domain containing, protein. The invention also features methods and compositions for modulation of the polypeptide and/or gene activity for prophylactic and therapeutic purposes, such as inhibition of progression of a cell to a metastatic cancerous cell.
It is a primary object of the invention to facilitate identification of cells that are of low metastatic potential (due to expression of GSEF) or high metastatic potential (due to absence of GSEF expression in a cell known to be cancerous).
One advantage of the invention is that detection of GSEF provides for sensitive and accurate detection of cells that are of high metastatic potential, and further provides for distinguishing high metastatic potential cells from low metastatic potential or non metastatic cells.
Additional objects and advantages will be readily apparent to the ordinarily skilled artisan upon reading the instant specification. Including a human GSEF promoter fragment specific for glandular epithelium of secretory glands (including prostate and breast) useful for gene theapie and the development of cancer mouse models.